Regulation of Flow-Induced K+ Wasting

描述（由申请人提供）：该项目的总体目标是阐明局部嘌呤能信号在调节晚期远端小管皮质集合管（CCD）的流量依赖性、不适当的K+分泌中的作用。众所周知，增强液体递送至晚期远端小管的状态诱导增强的K+分泌，这导致过量的K+排泄/K+消耗。这种流量依赖性K+消耗广泛发生在容量扩张、使用袢利尿剂和失盐性肾小管病变（如Bartter和Gitelman综合征）的情况下。它可以迅速导致低钾血症、血容量不足和低血压。虽然这种流动依赖性K+消耗的机制被认为涉及流动诱导的Ca 2+内流，其反过来激活晚期远端小管中的Ca 2+依赖性“BK”K+通道，但该机制仍然存在争议且知之甚少，特别是关于BK的Ca 2+敏感性以及该通道是否可以完全解释K+损失。我们最近确定了Ca 2+渗透性TRPV 4通道作为关键的流量敏感性Ca 2+内流途径，现在表明其流量依赖性在很大程度上受上游，流量敏感性，嘌呤能信号（局部ATP释放）耦合到PLC/DAG/PKC途径激活TRPV 4。重要的是，我们已经确定了一个新的Ca 2+依赖性K+通道，SK 3，具有比BK高得多的Ca 2+亲和力，它在CCD细胞的腔边界高度表达，并被流动激活。SK 3的激活使膜超极化，导致Ca 2+内流增强，我们假设这反过来支持低亲和力BK通道的激活。 我们的假设是，高肾小管流量激活TRPV 4（通过嘌呤能信号），TRPV 4介导的Ca 2+内流首先激活SK 3，增强Ca 2+内流，随后激活BK，导致流量诱导的K+分泌的两个K+通道。这项研究有两个目的：1）阐明SK 3和BK K+通道在CCD中的功能和相互依赖性，并阐明流动诱导的Ca 2+信号通过TRPV 4调节这些通道以引起流动敏感性K+分泌的机制，和2）通过对流量的关键评估来验证嘌呤能信号和增强的肾小管流量激活流量依赖性K+排泄的分子模型，敏感的信号成分在转基因动物模型的K+排泄。该项目创新性地使用细胞培养模型和天然的、分开的CCD来定义调控途径的关键方面（使用Ca 2+成像、电生理学、免疫荧光、生物化学/分子策略），并验证了K+排泄失调的转基因动物模型中的发现。这些研究的结果将为我们理解肾脏中流量敏感性K+排泄的分子基础提供新的见解，并将确定潜在的新治疗靶点，用于开发K+消耗性病理学的治疗策略。

DESCRIPTION (provided by applicant): The overall goal of the project is to elucidate the role of local purinergic signaling in regulating flow-dependent, inappropriate, K+ secretion by the cortical collecting ducts (CCD) of the late distal tubule. It is well known that states of enhance fluid delivery to the late distal tubule induces enhanced K+ secretion which results in excess K+ excretion/K+ wasting. Such flow-dependent K+ wasting is wide-spread occurring in conditions of volume expansion, loop-diuretic use and in salt-losing tubulopathies, such as Bartter and Gitelman syndromes. It can quickly lead to hypokalemia, volume depletion, and low blood pressure. While the mechanism of this flow- dependent K+ wasting is thought to involve flow-induced Ca2+ influx which, in turn, activates the Ca2+- dependent "BK" K+ channel in the late distal tubule, the mechanism remains controversial and poorly understood, especially with regard to the Ca2+ sensitivity of BK and whether this channel can fully account for the K+ lose. We recently identified the Ca2+-permeable TRPV4 channel as the key flow-sensitive Ca2+ influx pathway and now show that its flow-dependence is largely regulated by upstream, flow-sensitive, purinergic signaling (local ATP release) coupled to the PLC/DAG/PKC pathway to activate TRPV4. Importantly, we have identify a new Ca2+-dependent K+ channel, SK3, with a much higher Ca2+ affinity than BK, which is highly expressed at the luminal border of CCD cells and is activated by flow. Activation of SK3 hyperpolarizes the membrane leading to enhanced Ca2+ influx, which we postulate would, in turn, support activation of the low- affinity BK channel. Our hypothesis is that high tubular flow activates TRPV4 (via purinergic signaling) and that the TRPV4-mediated Ca2+ influx first activates SK3, enhancing Ca2+ influx, and subsequently activating BK leading to flow-induced K+ secretion by both K+ channels. The study has two aims: 1) To elucidate the function and interdependency of SK3 and BK K+ channels in CCD, and to elucidate the mechanism by which flow- induced Ca2+ signaling through TRPV4 regulates these channels to give rise to flow-sensitive K+ secretion, and 2) To verify the molecular model by which purinergic signaling and enhanced tubular flow activate flow- dependent K+ excretion by critical assessment of flow-sensitive signaling components in genetically modified animal models of K+ excretion. The project is innovative in the use of cell culture models and native, split- opened CCDs to define key aspects of the regulatory pathways (using Ca2+ imaging, electrophysiology, immunofluorescence, biochemical/molecular strategies), with verification of the findings in genetically modified animal models of dysregulate K+ excretion. The outcome of these studies will provide new insights into our understanding of the molecular basis of flow-sensitive K+ excretion in the kidney and will identify potential new therapeutic targets for development of treatment strategies in K+ wasting pathologies.